Currently, 800,000 patients receive chemotherapy in the United States each year. Myelosuppression and mucositis are common major side effects in conventional chemotherapy and are exacerbated by high dose and/or schedule intensification. As chemotherapeutic dosage has been identified as a major predictor of treatment outcome in a variety of cancer treatment programs, overcoming dose limiting toxicity has become a major goal for oncologists. Thus, to allow intensification of chemotherapy, it will be essential to prevent these unwanted side effects.
Studies have shown that both oral and gastrointestinal toxicity are a major problem in the clinic (Guggenheimer et al., 1977; Lockhart et al., 1981; Sonis et al., 1988; and Sonis & Clark, 1991). This toxicity is very common and has become dose or schedule limiting in both standard and high dose chemotherapy. As improvements are made in management of hematopoietic toxicity, oral toxicity is more frequently dose and schedule limiting. For example, 67% of leukemia/lymphoma patients, 75% of patients receiving bone marrow transplants following therapy, and approximately 20% of breast cancer patients, suffer from oral mucositis. It has been estimated that 40% of chemotherapy patients develop oral complications (Sonis et al., 1978). Many colon cancer patients receiving 5-fluorouracil (5-FU) suffer from oral mucositis.
Chemotherapy agents which cause oral and gastrointestinal mucositis include commonly prescribed agents. Single-agent or combination therapies which result in dose or schedule limiting oral and gastrointestinal (GI) toxicity include, but are not limited to, 5-FU, methotrexate, doxorubicin, Melphalan (see also Table A), and regimens such as the AFM regimen, the CEA regimen and the CAF regimen (Sonis et al., 1990). Cytotoxic agents, such as antiviral agents, also cause mucositis.
Oral mucositis following chemotherapy is a consequence of the high proliferative rate of the normal epithelial cells lining the oral cavity. Chemotherapy-induced mucositis in the oral cavity is largely derived from injury to the basal epithelial cell layer. The epithelial lining of the mouth is generally five cells thick, with the self-renewing stem cell layer located at the base, which itself overlays a fibrous connective tissue matrix. In contrast to skin, most of the epithelium which lines the oral cavity is not highly keratinized, and therefore is able to absorb and elicit a biological response to exogenously added peptide growth factors. The epithelium at the base of the mouth divides most rapidly and is most sensitive to chemotherapy.
The extent of oral mucositis appears dependent on the cycling status of the epithelial cell layer. For example, 90% of pediatric chemotherapy patients (ages 1-20) develop oral mucositis, as compared with 18% of patients over the age of sixty (Sonis et al., 1979). Further, in animal models of oral mucositis, increasing proliferation of the oral epithelium by prior administration of most growth factor (e.g. EGF, TGF-.alpha.) markedly increases the severity of oral mucositis. These positively acting factors are currently being evaluated for wound healing.
Oral mucositis leads to epithelial thinning and ulceration resulting in severe pain, weight loss from failure to eat or drink (often requiring parenteral feeding), infection (bacterial, fungal and viral), fever, nausea and diarrhea. Symptoms peak 7 to 10 days following therapy, and gradually recede over the following two weeks. Another major complication is potentially life-threatening infection due to sepsis. Therefore, methods of preventing oral toxicity would enable escalated schedules and doses of a chemotherapy regimen, resulting in improved patient long term survival for many human cancers, notably breast, lung and testicular cancer, leukemia, lymphoma, and neuroblastoma.
Chemotherapy-induced mucositis in the intestinal tract (gastrointestinal mucositis) is also marked and often dose limiting. In the development of the intestinal crypt, primitive stem cells in the proliferative zone at the base of the crypt give rise to more differentiated cell progeny which rapidly migrate to the top of the crypt. These more highly differentiated transitional cells have increasingly less capacity for self renewal and are eventually sloughed off at the top of the crypt. Cell proliferation is restricted to a centrally located band of cells (about ten cell layers deep) within the crypts. Within this band the cells divide rapidly (every ten to fourteen hours) and the product of this division activity matures and moves out of the crypt, onto the villus and is eventually lost within two to three days from the villus tip. Damage to the GI tract increases the risk of sepsis. Thus, like oral mucositis, there is a need to prevent gastrointestinal toxicity. TGF-.beta. is part of a family of multifunctional proteins which modulate, alone or in combination with other molecules, cell proliferation and differentiation. Mature TGF-.beta. is produced from a precursor form, consisting of a pro-region and mature TGF-.beta.. Known isoforms of mature TGF-.beta. are designated TGF-.beta.1, TGF-.beta.2, TGF-.beta.3, TGF-.beta.4, and TGF-.beta.5 (Brunner 1988). Mature TGF-.beta. has been isolated from various species (See EP 105,211 and U.S. Pat. No. 5,104,977). Across species TGF-.beta.s e.g. murine, bovine, human, and porcine show very little difference in amino acid composition. The expression of TGF-.beta. in both normal and transformed cells, and methods of producing biologically active mature TGF-.beta. from eucaryotic cells have been described (Madisen et al., 1988, Pincher et al., 1985, Assoian et al., 1983, Wrann et al., 1987).
TGF-.alpha. and TGF-.beta. are not homologous structures at the protein level and do not exhibit any sequence conservation at the nucleic acid level. As shown in Table 1 below, their physical and biological properties are also different.
TABLE 1 ______________________________________ COMPARISON OF PROPERTIES OF TGF-.alpha. and TGF-.beta. TGF-.alpha. TGF-.beta. ______________________________________ Physical Properties: Quaternary structure monomer dimer Subunit (amino acids) 50 112 Biological Properties: EGF receptor-binding + - Mitogenic activity in + - epithelial cells Arrests cell cycle in - + epithelial cells Stimulates fetal bone and - + collagen production ______________________________________
The ability of growth factors to accelerate healing is well known. Recently, Florine et al. U.S. Pat. No. 5,102,870, issued Apr. 7, 1992, described the use of positively acting growth factors and combinations to aid in the healing of oral mucositis. In contrast, TGF-.beta. is a negative regulator of epithelial cell division.
Myelosuppression has been a major dose-limiting toxicity in cancer chemotherapy, and antiviral therapy, e.g. AZT in AIDS therapy or management, which derives from the high proliferative rate of the hematopoietic compartment. The bone marrow contains a totipotent stem cell population that can give rise to a wide variety of cell types involved in oxygen transport, defense against infection. Granulocyte and neutrophil progenitor cells proliferate rapidly and their mature counterparts live only one to two days in the circulation. Consequently, 10-14 days after initiation of chemotherapy, the granulocyte and neutrophil population fall rapidly to a nadir. A reduction in megakaryocyte and erythroid precursor cell populations are also observed but this is less pronounced due to the longer life of their differentiated progeny. Thus, in many of the conventional protocols, chemotherapy is scheduled to maximize granulocyte/neutrophil recovery and to minimize infection.
Recent animal model data suggest that the ability to reconstitute the mature blood cell types decreases progressively with multiple rounds of chemotherapy, and administration of either G-CSF or GM-CSF accelerates the loss of self renewal capacity (Hornung and Longo, 1992). This phenomenon appears to result from progressive damage to the hematopoietic stem cell population. Although stem cells are normally quiescent, they are induced into active division following the first round of chemotherapy and thus become highly susceptible to the subsequent administration of cytotoxic drugs. Extensive experimentation has clearly demonstrated the phenomenon of stem cell depletion following multiple rounds of chemotherapy in animal models. This loss of hematopoietic renewal was one of the major driving forces behind the development of autologous bone marrow transplantation.
Amento et al. U.S. Pat. No. 5,108,989 issued April 28, 1992 discloses the systemic use of TGF-.beta. for predisposition of mammals to accelerated tissue repair. In contrast the present invention involves a method for inhibiting the cytotoxic poisoning of normal cells by slowing their growth.